Stable Formulations for Lyophilizing Therapeutic Particles

ABSTRACT

The present disclosure generally relates to lyophilized pharmaceutical compositions comprising polymeric nanoparticles which, upon reconstitution, have low levels of greater than 10 micron size particles. Other aspects of the invention include methods of making such nanoparticles.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/965,294, filed Dec. 10, 2010, which claims the benefit of andpriority to U.S. Provisional Patent Application No. 61/285,722, filedDec. 11, 2009, each of which is hereby incorporated by reference in itsentirety.

BACKGROUND

Systems that deliver certain drugs to a patient (e.g., targeted to aparticular tissue or cell type or targeted to a specific diseased tissuebut not normal tissue), or that control release of drugs has long beenrecognized as beneficial.

For example, therapeutics that include an active drug and that are,e.g., targeted to a particular tissue or cell type or targeted to aspecific diseased tissue but not to normal tissue, may reduce the amountof the drug in body tissues that do not require treatment. This isparticularly important when treating a condition such as cancer where itis desirable that a cytotoxic dose of the drug is delivered to cancercells without killing the surrounding non-cancerous tissue. Further,such therapeutics may reduce the undesirable and sometimeslife-threatening side effects common in anticancer therapy. In addition,such therapeutics may allow drugs to reach certain tissues they wouldotherwise be unable to reach.

Delivery of therapeutic nanoparticles can be achieved through parenteralinjection of a reconstituted suspension of the nanoparticles. Theoriginal nanoparticle suspension is lyophilized, i.e., freeze dried, forstorage before reconstitution. Freeze drying a nanoparticle suspensionpotentially creates a product for reconstitution with far superiorstorage stability than its frozen suspension counterpart. Further,freeze drying may provide easier storage that may not require constant,very low, temperatures. However, the reconstituted lyophilisate mustpossess physicochemical and performance attributes that are comparableor superior to the original suspension. Redispersing into particles ofthe same size without trace particulates due to micro-aggregation orundispersed particles is the most challenging aspect of nanoparticlesuspension lyophilization.

Accordingly, a need exists for nanoparticle therapeutics and methods ofmaking such nanoparticles, that are capable of delivering therapeuticlevels of drug to treat diseases such as cancer, and possess superiorstorage capabilities.

SUMMARY

In one aspect, the invention provides a lyophilized pharmaceuticalcomposition comprising polymeric nanoparticles, wherein uponreconstitution of the lyophilized pharmaceutical composition, in lessthan or about 100 mL of an aqueous medium, the reconstituted compositionsuitable for parenteral administration comprises: less than 6000microparticles of greater than or equal to 10 microns; and less than 600microparticles of greater than or equal to 25 microns. In oneembodiment, the reconstituted composition comprises less than 3000microparticles of greater than or equal to 10 microns; and less than 300microparticles of greater than or equal to 25 microns. In someembodiments, the nanoparticle concentration is about 50 mg/mL.

The number of microparticles can be determined by means such as the USP32 <788> by light obscuration particle count test, the USP 32<788> bymicroscopic particle count test, laser diffraction, and/or singleparticle optical sensing.

The nanoparticles may include an active agent or therapeutic agent,e.g., taxane, and one, two, or three biocompatible polymers. Forexample, disclosed herein is a therapeutic nanoparticle comprising about0.2 to about 35 weight percent of a therapeutic agent; about 10 to about99 weight percent poly(lactic) acid-block-poly(ethylene)glycol copolymeror poly(lactic)-co-poly(glycolic) acid-block-poly(ethylene)glycolcopolymer; and about 0 to about 50 weight percent poly(lactic) acid orpoly(lactic) acid-co-poly(glycolic) acid. Exemplary therapeutic agentsinclude antineoplastic agents such as taxanes, e.g., docetaxel and mayinclude about 10 to about 30 weight percent of a therapeutic agent,e.g., a taxane agent.

For example, the poly(lactic) acid portion of the copolymer may have aweight average molecular weight of about 16 kDa and thepoly(ethylene)glycol portion of the copolymer may have a weight averagemolecular weight of about 5 kDa.

Contemplated lyophilized pharmaceutical compositions may furthercomprise a sugar, e.g. a disaccharide, monosaccharide or polysaccharide,and an ionic halide salt. A disaccharide can be, for example, sucrose ortrehalose, or a mixture thereof. The ionic halide salt may be selectedfrom sodium chloride, calcium chloride, and zinc chloride, or mixturesthereof. In other embodiments, the lyophilized pharmaceuticalcomposition may also further comprise a cyclodextrin. For example, thelyophilized pharmaceutical composition may further comprise a sugar suchas a disaccharide, an ionic halide salt, and a cyclodextrin.Alternatively, the lyophilized pharmaceutical composition may furthercomprise a disaccharide and a cyclodextrin, and without the ionic halidesalt. The cyclodextrin may be selected from α-cyclodextrin,β-cyclodextrin, γ-cyclodextrin, or mixtures thereof.

The reconstituted composition may have minimal aggregation compared to areconstituted composition that does not contain an ionic halide saltand/or cyclodextrin. The reconstituted composition may have apolydispersity index of less than 0.2. In some embodiments, thenanoparticles have a concentration of about 10-100 mg/mL, e.g. 40-60mg/mL, or about 50 mg/mL.

In an aspect, the disclosure provides a pharmaceutical compositionsuitable for parenteral use upon reconstitution, comprising a pluralityof therapeutic particles each comprising a copolymer having ahydrophobic polymer segment and a hydrophilic polymer segment; an activeagent; a disaccharide; and an ionic halide salt and/or a cyclodextrinsuch as a beta-cyclodextrin (e.g. hydroxypropylcyclodextrin (HPbCD). Thepharmaceutical composition may further comprise a cyclodextrin. Inanother aspect, the disclosure provides a pharmaceutical compositionsuitable for parenteral use upon reconstitution, comprising a pluralityof therapeutic particles each comprising a copolymer having ahydrophobic polymer segment and a hydrophilic polymer segment; an activeagent; a disaccharide; and a cyclodextrin.

The ionic halide salt may be selected from the group consisting ofsodium chloride, calcium chloride, and zinc chloride, or mixturesthereof. The cyclodextrin may be selected from α-cyclodextrin,β-cyclodextrin, γ-cyclodextrin, or mixtures thereof. For example, thecopolymer may be poly(lactic) acid-block-poly(ethylene)glycol copolymer.Upon reconstitution, a 100 mL aqueous sample may comprise less than 6000particles having a size greater than or equal to 10 microns; and lessthan 600 particles having a size greater than or equal to 25 microns.

In another aspect, the invention provides a pharmaceutically acceptableformulation for parenteral administration, prepared by a processcomprising:

a) providing a composition comprising a plurality of therapeuticparticles each comprising a copolymer having a hydrophobic polymersegment and a hydrophilic polymer segment; and an active agent;

b) adding a disaccharide, an ionic halide salt, and optionally acyclodextrin to said composition;

c) lyophilizing the composition to form a lyophilized composition;

d) reconstituting the lyophilized composition to form the formulationsuitable for parenteral administration. The formulation may furthercomprise a cyclodextrin.

In yet another aspect, the invention provides a pharmaceuticallyacceptable formulation for parenteral administration, prepared by aprocess comprising:

a) providing a composition comprising a plurality of therapeuticparticles each comprising a copolymer having a hydrophobic polymersegment and a hydrophilic polymer segment; and an active agent;

b) adding a disaccharide and a cyclodextrin to said composition;

c) lyophilizing the composition to form a lyophilized composition;

d) reconstituting the lyophilized composition to form the formulationsuitable for parenteral administration.

The lyophilized composition may have a therapeutic particleconcentration of greater than about 40 mg/mL. The formulation suitablefor parenteral administration may have less than about 600 particleshaving a size greater than 10 microns in a 10 mL dose.

The step of adding a disaccharide and/or an ionic halide salt maycomprise adding about 5 to about 15 weight percent sucrose or about 5 toabout 20 weight percent (e.g. about 10 to about 20 weight percent)trehalose and about 10 to about 500 mM ionic halide salt. The step mayfurther comprise adding about 1 to about 25 weight percent cyclodextrin.

In another embodiment, the step of adding a disaccharide and acyclodextrin may comprise adding about 5 to about 15 weight percentsucrose or about 10 to about 20 weight percent trehalose and about 1 toabout 25 weight percent cyclodextrin.

The step of lyophilizing may comprise freezing the composition at atemperature of greater than about −40° C., or a temperature of less than−30° C., e.g. about −40° C. to about −30° C., or about −40° C. to about−25° C. forming a frozen composition; and drying the frozen compositionvia, e.g., sublimation, to form the lyophilized composition.

In another aspect, the disclosure provides a method of preventingsubstantial aggregation of particles in a pharmaceutical nanoparticlecomposition comprising adding a sugar and a salt to the lyophilizedformulation to prevent aggregation of the nanoparticles uponreconstitution. In an embodiment, cyclodextrin is also added to thelyophilized formulation. In yet another aspect, the disclosure providesa method of preventing substantial aggregation of particles in areconstituted pharmaceutical nanoparticle composition comprising addinga sugar and a cyclodextrin to a lyophilized formulation comprisingnanoparticles; reconstituting the lyophilized formulation, wherein thereconstituted composition does not have substantial aggregation of thenanoparticles. Also provided herein is a method of preventingsubstantial aggregation of particles in a pharmaceutical nanoparticlecomposition comprising adding a sugar and a cyclodextrin to thelyophilized formulation to prevent aggregation of the nanoparticles uponreconstitution.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart for an emulsion process for forming disclosednanoparticles.

FIGS. 2A and 2B are flow diagrams for a disclosed emulsion process. FIG.2A shows particle formation and hardening (upstream processing). FIG. 2Bshows particle work up and purification (downstream processing).

FIG. 3 depicts the effect of salt concentration and sucrose on particlesize in reconstituted nanoparticle suspensions.

FIG. 4 depicts temperature cycling for various lypholizationformulations.

FIG. 5 depicts the sizes via dynamic light scattering (DLS) of thevarious reconstituted nanoparticle suspensions disclosed herein.

FIG. 6 depicts the particulate counts of the various reconstitutednanoparticle suspensions disclosed herein

FIG. 7 depicts temperature cycling for various lypholizationformulations.

FIG. 8 depicts nanoparticle sizes (measured using DLS) of the variousreconstituted nanoparticle suspensions disclosed herein.

FIG. 9 depicts the particulate counts of the various reconstitutednanoparticle suspensions disclosed herein.

FIG. 10 depicts the particulate counts of the various reconstitutednanoparticle suspensions disclosed herein.

FIG. 11 depicts the particulate counts of the various reconstitutednanoparticle suspensions disclosed herein.

FIG. 12 depicts in vitro release of docetaxel of various nanoparticlesuspensions disclosed herein.

FIG. 13 depicts a differential scanning calorimetry (DSC) measurement ofnanoparticle suspensions having 5% trehalose and 10%hydroxypropylcyclodextrin.

FIG. 14 depicts the DSC properties of nanoparticle suspensions having10% trehalose and 10% hydroxypropylcyclodextrin.

FIG. 15 depicts the DSC properties of nanoparticle suspensions having20% trehalose and 15% hydroxypropylcyclodextrin.

FIG. 16 depicts the DSC properties of nanoparticle suspensions having10% sucrose and 10% hydroxypropylcyclodextrin.

DETAILED DESCRIPTION

The present invention generally relates to lyophilized polymericnanoparticle compositions, and methods of making and using suchtherapeutic compositions. Such compositions may be reconstituted from alyophilized composition, and may include minimal large aggregations ofnanoparticles and/or other materials. Disclosed compositions thereforemay be suitable for parenteral use.

Nanoparticles

In general, compositions may include nanoparticles that include anactive agent. As disclosed herein, “nanoparticle” refers to any particlehaving a diameter of less than 1000 nm, e.g., about 10 nm to about 200nm. Disclosed therapeutic nanoparticles may include nanoparticles havinga diameter of about 60 nm to about 120 nm, or about 70 nm to about 130nm, or about 60 nm to about 140 nm.

Disclosed nanoparticles may include about 0.2 to about 35 weightpercent, about 3 to about 40 weight percent, about 5 to about 30 weightpercent, 10 to about 30 weight percent, to 25 weight percent, or evenabout 4 to about 25 weight percent of an active agent, such as anantineoplastic agent, e.g., a taxane agent (for example, docetaxel).

Nanoparticles disclosed herein include one, two, three or morebiocompatible and/or biodegradable polymers. For example, a contemplatednanoparticle may include about 10 to about 99 weight percent of a one ormore block co-polymers that include a biodegradable polymer andpolyethylene glycol, and about 0 to about 50 weight percent of abiodegradable homopolymer.

Exemplary therapeutic nanoparticles may include about 40 to about 90weight percent poly(lactic) acid-poly(ethylene)glycol copolymer or about40 to about 80 weight percent poly(lactic) acid-poly(ethylene)glycolcopolymer. Such poly(lactic) acid-block-poly(ethylene)glycol copolymermay include poly(lactic acid) having a number average molecular weightof about 15 to 20 kDa (or for example about 15 to about 100 kDa, e.g.,about 15 to about 80 kDa), and poly(ethylene)glycol having a numberaverage molecular weight of about 2 to about 10 kDa, for example, about4 to about 6 kDa. For example, a disclosed therapeutic nanoparticle mayinclude about 70 to about 95 weight percent PLA-PEG and about 5 to about25 weight percent docetaxel. In another example, a disclosed therapeuticnanoparticle may include about 30 to about 50 weight percent PLA-PEG,about 30 to about 50 weight percent PLA or PLGA, and about 5 to about 25weight percent docetaxel. Such PLA ((poly)lactic acid) may have a numberaverage molecular weight of about 5 to about 10 kDa. Such PLGA (polylactic-co-glycolic acid) may have a number average molecular weight ofabout 8 to about 12 kDa.

In one embodiment, disclosed therapeutic nanoparticles may include atargeting ligand, e.g., a low-molecular weight PSMA ligand effective forthe treatment of a disease or disorder, such as prostate cancer, in asubject in need thereof. In certain embodiments, the low-molecularweight ligand is conjugated to a polymer, and the nanoparticle comprisesa certain ratio of ligand-conjugated polymer (e.g., PLA-PEG-Ligand) tonon-functionalized polymer (e.g., PLA-PEG or PLGA-PEG). The nanoparticlecan have an optimized ratio of these two polymers such that an effectiveamount of ligand is associated with the nanoparticle for treatment of adisease or disorder, such as cancer.

In some embodiments, disclosed nanoparticles may further comprise about0.2 to about 10 weight percent PLA-PEG functionalized with a targetingligand and/or may include about 0.2 to about 10 weight percent poly(lactic) acid-co poly (glycolic) acid block-PEG-functionalized with atargeting ligand. Such a targeting ligand may be, in some embodiments,covalently bound to the PEG, for example, bound to the PEG via analkylene linker, e.g., PLA-PEG-alkylene-GL2. For example, a disclosednanoparticle may include about 0.2 to about 10 mole percent PLA-PEG-GL2or poly (lactic) acid-co poly (glycolic) acid-PEG-GL2. It is understoodthat reference to PLA-PEG-GL2 or PLGA-PEG-GL2 refers to moieties thatmay include an alkylene linker (e.g., C₁-C₂₀, e.g., (CH₂)₅) linking thePEG to GL2.

In an embodiment, a therapeutic nanoparticle may include about 0.2 toabout 35 weight percent of a therapeutic agent; about 30 to about 99weight percent poly(lactic) acid-poly(ethylene)glycol copolymer orpoly(lactic)-co-poly (glycolic) acid-poly(ethylene)glycol copolymer;about 0 to about 50 weight percent poly(lactic) acid or poly(lactic)acid-co-poly (glycolic) acid; and about 0.2 to about 10 weight percent,or about 0.2 to about 30 weight percent PLA-PEG-GL2 or poly (lactic)acid-co poly (glycolic) acid-PEG-GL2. For example, PLA-PEG-GL2 mayinclude poly(lactic) acid with a number average molecular weight ofabout 10,000 Da to about 20,000 Da and poly(ethylene) glycol with anumber average molecular weight of about 4,000 to about 8,000.

Polymers

In some embodiments, the nanoparticles of the invention comprise amatrix of polymers and a therapeutic agent. In some embodiments, atherapeutic agent and/or targeting moiety (i.e., a low-molecular weightPSMA ligand) can be associated with at least part of the polymericmatrix. For example, in some embodiments, a targeting moiety (e.g.,ligand) can be covalently associated with the surface of a polymericmatrix. In some embodiments, covalent association is mediated by alinker. The therapeutic agent can be associated with the surface of,encapsulated within, surrounded by, and/or dispersed throughout thepolymeric matrix.

A wide variety of polymers and methods for forming particles therefromare known in the art of drug delivery. In some embodiments, thedisclosure is directed toward nanoparticles with at least twomacromolecules, wherein the first macromolecule comprises a firstpolymer bound to a low-molecular weight ligand (e.g., targeting moiety);and the second macromolecule comprising a second polymer that is notbound to a targeting moiety. The nanoparticle can optionally include oneor more additional, unfunctionalized, polymers.

Any polymer can be used in accordance with the present invention.Polymers can be natural or unnatural (synthetic) polymers. Polymers canbe homopolymers or copolymers comprising two or more monomers. In termsof sequence, copolymers can be random, block, or comprise a combinationof random and block sequences. Typically, polymers in accordance withthe present invention are organic polymers.

The term “polymer,” as used herein, is given its ordinary meaning asused in the art, i.e., a molecular structure comprising one or morerepeat units (monomers), connected by covalent bonds. The repeat unitsmay all be identical, or in some cases, there may be more than one typeof repeat unit present within the polymer. In some cases, the polymercan be biologically derived, i.e., a biopolymer. Non-limiting examplesinclude peptides or proteins. In some cases, additional moieties mayalso be present in the polymer, for example biological moieties such asthose described below. If more than one type of repeat unit is presentwithin the polymer, then the polymer is said to be a “copolymer.” It isto be understood that in any embodiment employing a polymer, the polymerbeing employed may be a copolymer in some cases. The repeating unitsforming the copolymer may be arranged in any fashion. For example, therepeating units may be arranged in a random order, in an alternatingorder, or as a block copolymer, i.e., comprising one or more regionseach comprising a first repeat unit (e.g., a first block), and one ormore regions each comprising a second repeat unit (e.g., a secondblock), etc. Block copolymers may have two (a diblock copolymer), three(a triblock copolymer), or more numbers of distinct blocks.

Disclosed particles can include copolymers, which, in some embodiments,describes two or more polymers (such as those described herein) thathave been associated with each other, usually by covalent bonding of thetwo or more polymers together. Thus, a copolymer may comprise a firstpolymer and a second polymer, which have been conjugated together toform a block copolymer where the first polymer can be a first block ofthe block copolymer and the second polymer can be a second block of theblock copolymer. Of course, those of ordinary skill in the art willunderstand that a block copolymer may, in some cases, contain multipleblocks of polymer, and that a “block copolymer,” as used herein, is notlimited to only block copolymers having only a single first block and asingle second block. For instance, a block copolymer may comprise afirst block comprising a first polymer, a second block comprising asecond polymer, and a third block comprising a third polymer or thefirst polymer, etc. In some cases, block copolymers can contain anynumber of first blocks of a first polymer and second blocks of a secondpolymer (and in certain cases, third blocks, fourth blocks, etc.). Inaddition, it should be noted that block copolymers can also be formed,in some instances, from other block copolymers. For example, a firstblock copolymer may be conjugated to another polymer (which may be ahomopolymer, a biopolymer, another block copolymer, etc.), to form a newblock copolymer containing multiple types of blocks, and/or to othermoieties (e.g., to non-polymeric moieties).

In some embodiments, the polymer (e.g., copolymer, e.g., blockcopolymer) can be amphiphilic, i.e., having a hydrophilic portion and ahydrophobic portion, or a relatively hydrophilic portion and arelatively hydrophobic portion. A hydrophilic polymer can be onegenerally that attracts water and a hydrophobic polymer can be one thatgenerally repels water. A hydrophilic or a hydrophobic polymer can beidentified, for example, by preparing a sample of the polymer andmeasuring its contact angle with water (typically, the polymer will havea contact angle of less than 60°, while a hydrophobic polymer will havea contact angle of greater than about 60°). In some cases, thehydrophilicity of two or more polymers may be measured relative to eachother, i.e., a first polymer may be more hydrophilic than a secondpolymer. For instance, the first polymer may have a smaller contactangle than the second polymer.

In one set of embodiments, a polymer (e.g., copolymer, e.g., blockcopolymer) contemplated herein includes a biocompatible polymer, i.e.,the polymer that does not typically induce an adverse response wheninserted or injected into a living subject, for example, withoutsignificant inflammation and/or acute rejection of the polymer by theimmune system, for instance, via a T-cell response. Accordingly, thetherapeutic particles contemplated herein can be non-immunogenic. Theterm non-immunogenic as used herein refers to endogenous growth factorin its native state which normally elicits no, or only minimal levelsof, circulating antibodies, T-cells, or reactive immune cells, and whichnormally does not elicit in the individual an immune response againstitself.

Biocompatibility typically refers to the acute rejection of material byat least a portion of the immune system, i.e., a nonbiocompatiblematerial implanted into a subject provokes an immune response in thesubject that can be severe enough such that the rejection of thematerial by the immune system cannot be adequately controlled, and oftenis of a degree such that the material must be removed from the subject.One simple test to determine biocompatibility can be to expose a polymerto cells in vitro; biocompatible polymers are polymers that typicallywill not result in significant cell death at moderate concentrations,e.g., at concentrations of 50 micrograms/10⁶ cells. For instance, abiocompatible polymer may cause less than about 20% cell death whenexposed to cells such as fibroblasts or epithelial cells, even ifphagocytosed or otherwise uptaken by such cells. Non-limiting examplesof biocompatible polymers that may be useful in various embodiments ofthe present invention include polydioxanone (PDO), polyhydroxyalkanoate,polyhydroxybutyrate, poly(glycerol sebacate), polyglycolide,polylactide, PLGA, polycaprolactone, or copolymers or derivativesincluding these and/or other polymers.

In certain embodiments, contemplated biocompatible polymers may bebiodegradable, i.e., the polymer is able to degrade, chemically and/orbiologically, within a physiological environment, such as within thebody. As used herein, “biodegradable” polymers are those that, whenintroduced into cells, are broken down by the cellular machinery(biologically degradable) and/or by a chemical process, such ashydrolysis, (chemically degradable) into components that the cells caneither reuse or dispose of without significant toxic effect on thecells. In one embodiment, the biodegradable polymer and theirdegradation byproducts can be biocompatible.

For instance, a contemplated polymer may be one that hydrolyzesspontaneously upon exposure to water (e.g., within a subject), thepolymer may degrade upon exposure to heat (e.g., at temperatures ofabout 37° C.). Degradation of a polymer may occur at varying rates,depending on the polymer or copolymer used. For example, the half-lifeof the polymer (the time at which 50% of the polymer can be degradedinto monomers and/or other nonpolymeric moieties) may be on the order ofdays, weeks, months, or years, depending on the polymer. The polymersmay be biologically degraded, e.g., by enzymatic activity or cellularmachinery, in some cases, for example, through exposure to a lysozyme(e.g., having relatively low pH). In some cases, the polymers may bebroken down into monomers and/or other nonpolymeric moieties that cellscan either reuse or dispose of without significant toxic effect on thecells (for example, polylactide may be hydrolyzed to form lactic acid,polyglycolide may be hydrolyzed to form glycolic acid, etc.).

In some embodiments, polymers may be polyesters, including copolymerscomprising lactic acid and glycolic acid units, such as poly(lacticacid-co-glycolic acid) and poly(lactide-co-glycolide), collectivelyreferred to herein as “PLGA”; and homopolymers comprising glycolic acidunits, referred to herein as “PGA,” and lactic acid units, such aspoly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid,poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectivelyreferred to herein as “PLA.” In some embodiments, exemplary polyestersinclude, for example, polyhydroxyacids; PEGylated polymers andcopolymers of lactide and glycolide (e.g., PEGylated PLA, PEGylated PGA,PEGylated PLGA, and derivatives thereof. In some embodiments, polyestersinclude, for example, polyanhydrides, poly(ortho ester) PEGylatedpoly(ortho ester), poly(caprolactone), PEGylated poly(caprolactone),polylysine, PEGylated polylysine, poly(ethylene imine), PEGylatedpoly(ethylene imine), poly(L-lactide-co-L-lysine), poly(serine ester),poly(4-hydroxy-L-proline ester), poly[α-(4-aminobutyl)-L-glycolic acid],and derivatives thereof.

In some embodiments, a polymer may be PLGA. PLGA is a biocompatible andbiodegradable co-polymer of lactic acid and glycolic acid, and variousforms of PLGA can be characterized by the ratio of lactic acid:glycolicacid. Lactic acid can be L-lactic acid, D-lactic acid, or D,L-lacticacid. The degradation rate of PLGA can be adjusted by altering thelactic acid-glycolic acid ratio. In some embodiments, PLGA to be used inaccordance with the present invention can be characterized by a lacticacid:glycolic acid ratio of approximately 85:15, approximately 75:25,approximately 60:40, approximately 50:50, approximately 40:60,approximately 25:75, or approximately 15:85. In some embodiments, theratio of lactic acid to glycolic acid monomers in the polymer of theparticle (e.g., the PLGA block copolymer or PLGA-PEG block copolymer),may be selected to optimize for various parameters such as water uptake,therapeutic agent release and/or polymer degradation kinetics can beoptimized.

In some embodiments, polymers may be one or more acrylic polymers. Incertain embodiments, acrylic polymers include, for example, acrylic acidand methacrylic acid copolymers, methyl methacrylate copolymers,ethoxyethyl methacrylates, cyanoethyl methacrylate, amino alkylmethacrylate copolymer, poly(acrylic acid), poly(methacrylic acid),methacrylic acid alkylamide copolymer, poly(methyl methacrylate),poly(methacrylic acid polyacrylamide, amino alkyl methacrylatecopolymer, glycidyl methacrylate copolymers, polycyanoacrylates, andcombinations comprising one or more of the foregoing polymers. Theacrylic polymer may comprise fully-polymerized copolymers of acrylic andmethacrylic acid esters with a low content of quaternary ammoniumgroups.

In some embodiments, polymers can be cationic polymers. In general,cationic polymers are able to condense and/or protect negatively chargedstrands of nucleic acids (e.g. DNA, RNA, or derivatives thereof).Amine-containing polymers such as poly(lysine), polyethylene imine(PEI), and poly(amidoamine) dendrimers are contemplated for use, in someembodiments, in a disclosed particle.

In some embodiments, polymers can be degradable polyesters bearingcationic side chains. Examples of these polyesters includepoly(L-lactide-co-L-lysine), poly(serine ester),poly(4-hydroxy-L-proline ester).

Particles disclosed herein may or may not contain PEG. In addition,certain embodiments can be directed towards copolymers containingpoly(ester-ether)s, e.g., polymers having repeat units joined by esterbonds (e.g., R—C(O)—O—R′ bonds) and ether bonds (e.g., R—O—R′ bonds). Insome embodiments of the invention, a biodegradable polymer, such as ahydrolyzable polymer, containing carboxylic acid groups, may beconjugated with poly(ethylene glycol) repeat units to form apoly(ester-ether). A polymer (e.g., copolymer, e.g., block copolymer)containing poly(ethylene glycol) repeat units can also be referred to asa “PEGylated” polymer.

It is contemplated that PEG may be terminated and include an end group,for example, when PEG is not conjugated to a ligand. For example, PEGmay terminate in a hydroxyl, a methoxy or other alkoxyl group, a methylor other alkyl group, an aryl group, a carboxylic acid, an amine, anamide, an acetyl group, a guanidino group, or an imidazole. Othercontemplated end groups include azide, alkyne, maleimide, aldehyde,hydrazide, hydroxylamine, alkoxyamine, or thiol moieties.

Those of ordinary skill in the art will know of methods and techniquesfor PEGylating a polymer, for example, by using EDC(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) and NHS(N-hydroxysuccinimide) to react a polymer to a PEG group terminating inan amine, by ring opening polymerization techniques (ROMP), or the like

In one embodiment, the molecular weight of the polymers can be optimizedfor effective treatment as disclosed herein. For example, the molecularweight of a polymer may influence particle degradation rate (such aswhen the molecular weight of a biodegradable polymer can be adjusted),solubility, water uptake, and drug release kinetics. For example, themolecular weight of the polymer can be adjusted such that the particlebiodegrades in the subject being treated within a reasonable period oftime (ranging from a few hours to 1-2 weeks, 3-4 weeks, 5-6 weeks, 7-8weeks, etc.). A disclosed particle can for example comprise a diblockcopolymer of PEG and PL(G)A, wherein for example, the PEG portion mayhave a number average molecular weight of about 1,000-20,000, e.g.,about 2,000-20,000, e.g., about 2 to about 10,000, and the PL(G)Aportion may have a number average molecular weight of about 5,000 toabout 20,000, or about 5,000-100,000, e.g., about 20,000-70,000, e.g.,about 15,000-50,000.

For example, disclosed here is an exemplary therapeutic nanoparticlethat includes about 10 to about 99 weight percent poly(lactic)acid-poly(ethylene)glycol copolymer or poly(lactic)-co-poly (glycolic)acid-poly(ethylene)glycol copolymer, or about 20 to about 80 weightpercent, about 40 to about 80 weight percent, or about 30 to about 50weight percent, or about 70 to about 90 weight percent poly(lactic)acid-poly(ethylene)glycol copolymer or poly(lactic)-co-poly (glycolic)acid-poly(ethylene)glycol copolymer. Exemplary poly(lactic)acid-poly(ethylene)glycol copolymers can include a number averagemolecular weight of about 15 to about 20 kDa, or about 10 to about 25kDa of poly(lactic) acid and a number average molecular weight of about4 to about 6, or about 2 kDa to about 10 kDa of poly(ethylene)glycol.

Disclosed nanoparticles may optionally include about 1 to about 50weight percent poly(lactic) acid or poly(lactic) acid-co-poly (glycolic)acid (which does not include PEG), or may optionally include about 1 toabout 50 weight percent, or about 10 to about 50 weight percent or about30 to about 50 weight percent poly(lactic) acid or poly(lactic)acid-co-poly (glycolic) acid. For example, poly(lactic) orpoly(lactic)-co-poly(glycolic) acid may have a number average moleculeweight of about 5 to about 15 kDa, or about 5 to about 12 kDa. ExemplaryPLA may have a number average molecular weight of about 5 to about 10kDa. Exemplary PLGA may have a number average molecular weight of about8 to about 12 kDa.

In certain embodiments, the polymers of the nanoparticles can beconjugated to a lipid. The polymer can be, for example, alipid-terminated PEG. As described below, the lipid portion of thepolymer can be used for self assembly with another polymer, facilitatingthe formation of a nanoparticle. For example, a hydrophilic polymercould be conjugated to a lipid that will self assemble with ahydrophobic polymer.

In some embodiments, lipids are oils. In general, any oil known in theart can be conjugated to the polymers used in the invention. In someembodiments, an oil can comprise one or more fatty acid groups or saltsthereof. In some embodiments, a fatty acid group can comprisedigestible, long chain (e.g., C₈-C₅₀), substituted or unsubstitutedhydrocarbons. In some embodiments, a fatty acid group can be a C₁₀-C₂₀fatty acid or salt thereof. In some embodiments, a fatty acid group canbe a C₁₅-C₂₀ fatty acid or salt thereof. In some embodiments, a fattyacid can be unsaturated. In some embodiments, a fatty acid group can bemonounsaturated. In some embodiments, a fatty acid group can bepolyunsaturated. In some embodiments, a double bond of an unsaturatedfatty acid group can be in the cis conformation. In some embodiments, adouble bond of an unsaturated fatty acid can be in the transconformation.

In some embodiments, a fatty acid group can be one or more of butyric,caproic, caprylic, capric, lauric, myristic, palmitic, stearic,arachidic, behenic, or lignoceric acid. In some embodiments, a fattyacid group can be one or more of palmitoleic, oleic, vaccenic, linoleic,alpha-linolenic, gamma-linoleic, arachidonic, gadoleic, arachidonic,eicosapentaenoic, docosahexaenoic, or erucic acid.

In one embodiment, optional small molecule targeting moieties arebonded, e.g., covalently bonded, to the lipid component of thenanoparticle. For example, provided herein is a nanoparticle comprisinga therapeutic agent, a polymeric matrix comprising functionalized andnon-functionalized polymers, a lipid, and a low-molecular weight PSMAtargeting ligand, wherein the targeting ligand is bonded, e.g.,covalently bonded, to the lipid component of the nanoparticle. In oneembodiment, the lipid component that is bonded to the low-molecularweight targeting moiety is of the Formula V:

and salts thereof, wherein each R is, independently, C₁₋₃₀ alkyl. In oneembodiment of Formula V, the lipid can be 1,2distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and salts thereof,e.g., the sodium salt. In another embodiment, the invention provides atarget-specific nanoparticle comprising a therapeutic agent, a polymericmatrix, DSPE, and a low-molecular weight PSMA targeting ligand, whereinthe ligand is bonded, e.g., covalently bonded, to DSPE. For example, thenanoparticle of the invention may comprise a polymeric matrix comprisingPLGA-DSPE-PEG-Ligand.

A contemplated nanoparticle may include a ratio of ligand-bound polymerto non-functionalized polymer effective for the treatment of prostatecancer, wherein the hydrophilic, ligand-bound polymer is conjugated to alipid that will self assemble with the hydrophobic polymer, such thatthe hydrophobic and hydrophilic polymers that constitute thenanoparticle are not covalently bound. “Self-assembly” refers to aprocess of spontaneous assembly of a higher order structure that relieson the natural attraction of the components of the higher orderstructure (e.g., molecules) for each other. It typically occurs throughrandom movements of the molecules and formation of bonds based on size,shape, composition, or chemical properties. For example, such a methodcomprises providing a first polymer that is reacted with a lipid, toform a polymer/lipid conjugate. The polymer/lipid conjugate is thenreacted with the low-molecular weight ligand to prepare a ligand-boundpolymer/lipid conjugate; and mixing the ligand-bound polymer/lipidconjugate with a second, non-functionalized polymer, and the therapeuticagent; such that the nanoparticle is formed. In certain embodiments, thefirst polymer is PEG, such that a lipid-terminated PEG is formed. In oneembodiment, the lipid is of the Formula V, e.g., 2distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and salts thereof,e.g., the sodium salt. The lipid-terminated PEG can then, for example,be mixed with PLGA to form a nanoparticle.

Targeting Moieties

Provided herein are nanoparticles that may include an optional targetingmoiety, i.e., a moiety able to bind to or otherwise associate with abiological entity, for example, a membrane component, a cell surfacereceptor, prostate specific membrane antigen, or the like. A targetingmoiety present on the surface of the particle may allow the particle tobecome localized at a particular targeting site, for instance, a tumor,a disease site, a tissue, an organ, a type of cell, etc. As such, thenanoparticle may then be “target specific.” The drug or other payloadmay then, in some cases, be released from the particle and allowed tointeract locally with the particular targeting site.

In a particular embodiment, the drug or other payload may be released ina controlled release manner from the particle and allowed to interactlocally with the particular targeting site (e.g., a tumor). The term“controlled release” (and variants of that term) as used herein (e.g.,in the context of “controlled-release system”) is generally meant toencompass release of a substance (e.g., a drug) at a selected site orotherwise controllable in rate, interval, and/or amount. Controlledrelease encompasses, but is not necessarily limited to, substantiallycontinuous delivery, patterned delivery (e.g., intermittent deliveryover a period of time that is interrupted by regular or irregular timeintervals), and delivery of a bolus of a selected substance (e.g., as apredetermined, discrete amount if a substance over a relatively shortperiod of time (e.g., a few seconds or minutes)).

In one embodiment, a disclosed nanoparticle includes a targeting moietythat is a low-molecular weight ligand, e.g., a low-molecular weight PSMAligand. The term “bind” or “binding,” as used herein, refers to theinteraction between a corresponding pair of molecules or portionsthereof that exhibit mutual affinity or binding capacity, typically dueto specific or non-specific binding or interaction, including, but notlimited to, biochemical, physiological, and/or chemical interactions.“Biological binding” defines a type of interaction that occurs betweenpairs of molecules including proteins, nucleic acids, glycoproteins,carbohydrates, hormones, or the like. The term “binding partner” refersto a molecule that can undergo binding with a particular molecule.“Specific binding” refers to molecules, such as polynucleotides, thatare able to bind to or recognize a binding partner (or a limited numberof binding partners) to a substantially higher degree than to other,similar biological entities. In one set of embodiments, the targetingmoiety has an affinity (as measured via a disassociation constant) ofless than about 1 micromolar, at least about 10 micromolar, or at leastabout 100 micromolar.

For example, a targeting portion may cause the particles to becomelocalized to a tumor (e.g., a solid tumor) a disease site, a tissue, anorgan, a type of cell, etc. within the body of a subject, depending onthe targeting moiety used. For example, a low-molecular weight PSMAligand may become localized to a solid tumor, e.g., breast or prostatetumors or cancer cells. The subject may be a human or non-human animal.Examples of subjects include, but are not limited to, a mammal such as adog, a cat, a horse, a donkey, a rabbit, a cow, a pig, a sheep, a goat,a rat, a mouse, a guinea pig, a hamster, a primate, a human or the like.

For example, a targeting moiety may small target prostate cancer tumors,for example a target moiety may be PSMA peptidase inhibitor. Thesemoieties are also referred to herein as “low-molecular weight PSMAligands.” When compared with expression in normal tissues, expression ofprostate specific membrane antigen (PSMA) is at least 10-foldoverexpressed in malignant prostate relative to normal tissue, and thelevel of PSMA expression is further up-regulated as the diseaseprogresses into metastatic phases (Silver et al. 1997, Clin. CancerRes., 3:81).

For example, the low-molecular weight PSMA ligand is

and enantiomers, stereoisomers, rotamers, tautomers, diastereomers, orracemates thereof. Particularly, the butyl-amine compound has theadvantage of ease of synthesis, especially because of its lack of abenzene ring.

For example, a disclosed nanoparticle may include a conjugaterepresented by:

where y is about 222 and z is about 114.

For example, a disclosed nanoparticle includes a polymeric compoundselected from:

wherein R₁ is selected from the group consisting of H, and a C₁-C₂₀alkyl group optionally substituted with halogen;

R₂ is a bond, an ester linkage, or amide linkage;

R₃ is an C₁-C₁₀ alkylene or a bond;

x is 50 to about 1500, for example about 170 to about 260;

y is 0 to about 50, for example y is 0; and

z is about 30 to about 456, or about 30 to about 200, for example, z isabout 80 to about 130.

Therapeutic Agents

Agents including, for example, therapeutic agents (e.g., anti-canceragents), diagnostic agents (e.g., contrast agents; radionuclides; andfluorescent, luminescent, and magnetic moieties), prophylactic agents(e.g., vaccines), and/or nutraceutical agents (e.g., vitamins, minerals,etc.) compose part of the disclosed nanoparticles. Exemplary agents tobe delivered in accordance with the present invention include, but arenot limited to, small molecules (e.g., cytotoxic agents), nucleic acids(e.g., siRNA, RNAi, and microRNA agents), proteins (e.g., antibodies),peptides, lipids, carbohydrates, hormones, metals, radioactive elementsand compounds, drugs, vaccines, immunological agents, etc., and/orcombinations thereof. In some embodiments, the agent to be delivered isan agent useful in the treatment of cancer (e.g., prostate cancer).

The active agent or drug may be a therapeutic agent (e.g. achemotherapeutic) such as mTor inhibitors (e.g., sirolimus,temsirolimus, or everolimus), vinca alkaloids (e.g. vinorelbine orvincristine), a diterpene derivative, a taxane (e.g. paclitaxel or itsderivatives such as DHA-paclitaxel or PG-paclitaxel, or docetaxel),), acardiovascular agent (e.g. a diuretic, a vasodilator, angiotensinconverting enzyme, a beta blocker, an aldosterone antagonist, or a bloodthinner), a corticosteroid, an antimetabolite or antifolate agent (e.g.methotrexate), a chemotherapeutic agent (e.g. epothilone B), analkylating agent (e.g. bendamustine), or the active agent or drug may bean siRNA.

In an embodiment, an active or therapeutic agent may (or may not be)conjugated to e.g. a disclosed polymer that forms part of a disclosednanoparticle, e.g. an active agent may be conjugated (e.g. covalentlybound, e.g. directly or through a linking moiety) to PLA or PGLA, or aPLA or PLGA portion of a copolymer such as PLA-PEG or PLGA-PEG

Preparation of Nanoparticles

Another aspect of this disclosure is directed to systems and methods ofmaking disclosed nanoparticles. In some embodiments, using two or moredifferent polymers (e.g., copolymers, e.g., block copolymers) indifferent ratios and producing particles from the polymers (e.g.,copolymers, e.g., block copolymers), properties of the particles becontrolled. For example, one polymer (e.g., copolymer, e.g., blockcopolymer) may include a low-molecular weight PSMA ligand, while anotherpolymer (e.g., copolymer, e.g., block copolymer) may be chosen for itsbiocompatibility and/or its ability to control immunogenicity of theresultant particle.

In one set of embodiments, the particles are formed by providing asolution comprising one or more polymers, and contacting the solutionwith a polymer nonsolvent to produce the particle. The solution may bemiscible or immiscible with the polymer nonsolvent. For example, awater-miscible liquid such as acetonitrile may contain the polymers, andparticles are formed as the acetonitrile is contacted with water, apolymer nonsolvent, e.g., by pouring the acetonitrile into the water ata controlled rate. The polymer contained within the solution, uponcontact with the polymer nonsolvent, may then precipitate to formparticles such as nanoparticles. Two liquids are said to be “immiscible”or not miscible, with each other when one is not soluble in the other toa level of at least 10% by weight at ambient temperature and pressure.Typically, an organic solution (e.g., dichloromethane, acetonitrile,chloroform, tetrahydrofuran, acetone, formamide, dimethylformamide,pyridines, dioxane, dimethysulfoxide, etc.) and an aqueous liquid (e.g.,water, or water containing dissolved salts or other species, cell orbiological media, ethanol, etc.) are immiscible with respect to eachother. For example, the first solution may be poured into the secondsolution (at a suitable rate or speed). In some cases, particles such asnanoparticles may be formed as the first solution contacts theimmiscible second liquid, e.g., precipitation of the polymer uponcontact causes the polymer to form nanoparticles while the firstsolution is poured into the second liquid, and in some cases, forexample, when the rate of introduction is carefully controlled and keptat a relatively slow rate, nanoparticles may form. The control of suchparticle formation can be readily optimized by one of ordinary skill inthe art using only routine experimentation.

In another embodiment, a nanoemulsion process is provided, such as theprocess represented in FIGS. 1, 2A, and 2B. For example, a therapeuticagent, a first polymer (for example, a diblock co-polymer such asPLA-PEG or PLGA-PEG, either of which may be optionally bound to aligand, e.g., GL2) and an optional second polymer (e.g., (PL(G)A-PEG orPLA), with an organic solution to form a first organic phase. Such firstphase may include about 5 to about 50% weight solids, e.g. about 5 toabout 40% solids, or about 10 to about 30% solids. The first organicphase may be combined with a first aqueous solution to form a secondphase. The organic solution can include, for example, toluene, methylethyl ketone, acetonitrile, tetrahydrofuran, ethyl acetate, isopropylalcohol, isopropyl acetate, dimethylformamide, methylene chloride,dichloromethane, chloroform, acetone, benzyl alcohol, Tween 80, Span 80,or the like, and combinations thereof. In an embodiment, the organicphase may include benzyl alcohol, ethyl acetate, and combinationsthereof. The second phase can be between about 1 and 50 weight percent,e.g., about 5-40 weight percent, solids. The aqueous solution can bewater, optionally in combination with one or more of sodium cholate,ethyl acetate, polyvinyl acetate and benzyl alcohol.

For example, the oil or organic phase may use solvent that is onlypartially miscible with the nonsolvent (water). Therefore, when mixed ata low enough ratio and/or when using water pre-saturated with theorganic solvents, the oil phase remains liquid. The oil phase may beemulsified into an aqueous solution and, as liquid droplets, shearedinto nanoparticles using, for example, high energy dispersion systems,such as homogenizers or sonicators. The aqueous portion of the emulsion,otherwise known as the “water phase”, may be a surfactant solutionconsisting of sodium cholate and pre-saturated with ethyl acetate andbenzyl alcohol.

Emulsifying the second phase to form an emulsion phase may be performedin one or two emulsification steps. For example, a primary emulsion maybe prepared, and then emulsified to form a fine emulsion. The primaryemulsion can be formed, for example, using simple mixing, a highpressure homogenizer, probe sonicator, stir bar, or a rotor statorhomogenizer. The primary emulsion may be formed into a fine emulsionthrough the use of e.g., probe sonicator or a high pressure homogenizer,e.g., by using 1, 2, 3 or more passes through a homogenizer. Forexample, when a high pressure homogenizer is used, the pressure used maybe about 1000 to about 8000 psi, about 2000 to about 4000 psi4000 toabout 8000 psi, or about 4000 to about 5000 psi, e.g., about 2000, 2500,4000 or 5000 psi.

Either solvent evaporation or dilution may be needed to complete theextraction of the solvent and solidify the particles. For better controlover the kinetics of extraction and a more scalable process, a solventdilution via aqueous quench may be used. For example, the emulsion canbe diluted into cold water to a concentration sufficient to dissolve allof the organic solvent to form a quenched phase. Quenching may beperformed at least partially at a temperature of about 5° C. or less.For example, water used in the quenching may be at a temperature that isless that room temperature (e.g., about 0 to about 10° C., or about 0 toabout 5° C.).

In some embodiments, not all of the therapeutic agent (e.g., docetaxel)is encapsulated in the particles at this stage, and a drug solubilizeris added to the quenched phase to form a solubilized phase. The drugsolubilizer may be for example, Tween 80, Tween 20, polyvinylpyrrolidone, cyclodextran, sodium dodecyl sulfate, or sodium cholate.For example, Tween-80 may added to the quenched nanoparticle suspensionto solubilize the free drug and prevent the formation of drug crystals.In some embodiments, a ratio of drug solubilizer to therapeutic agent(e.g., docetaxel) is about 100:1 to about 10:1.

The solubilized phase may be filtered to recover the nanoparticles. Forexample, ultrafiltration membranes may be used to concentrate thenanoparticle suspension and substantially eliminate organic solvent,free drug, and other processing aids (surfactants). Exemplary filtrationmay be performed using a tangential flow filtration system. For example,by using a membrane with a pore size suitable to retain nanoparticleswhile allowing solutes, micelles, and organic solvent to pass,nanoparticles can be selectively separated. Exemplary membranes withmolecular weight cut-offs of about 300-500 kDa (−5-25 nm) may be used.

Diafiltration may be performed using a constant volume approach, meaningthe diafiltrate (cold deionized water, e.g., about 0 to about 5° C., or0 to about 10° C.) may added to the feed suspension at the same rate asthe filtrate is removed from the suspension. In some embodiments,filtering may include a first filtering using a first temperature ofabout 0 to about 5° C., or 0 to about 10° C., and a second temperatureof about 20 to about 30° C., or 15 to about 35° C. For example,filtering may include processing about 1 to about 6 diavolumes at about0 to about 5° C., and processing at least one diavolume (e.g., about 1to about 3 or about 1-2 diavolumes) at about 20 to about 30° C.

After purifying and concentrating the nanoparticle suspension, theparticles may be passed through one, two or more sterilizing and/ordepth filters, for example, using ˜0.2 μm depth pre-filter.

In another embodiment of preparing nanoparticles, an organic phase isformed composed of a mixture of a therapeutic agent, e.g., docetaxel,and polymer (homopolymer, co-polymer, and co-polymer with ligand). Theorganic phase is mixed with an aqueous phase at approximately a 1:5ratio (oil phase:aqueous phase) where the aqueous phase is composed of asurfactant and some dissolved solvent. The primary emulsion is formed bythe combination of the two phases under simple mixing or through the useof a rotor stator homogenizer. The primary emulsion is then formed intoa fine emulsion through the use of a high pressure homogenizer. The fineemulsion is then quenched by addition to deionized water under mixing.The quench:emulsion ratio is approximately 8.5:1. Then a solution ofTween (e.g., Tween 80) is added to the quench to achieve approximately2% Tween overall. This serves to dissolve free, unencapsulated drug. Thenanoparticles are then isolated through either centrifugation orultrafiltration/diafiltration.

It will be appreciated that the amounts of polymer and therapeutic oractive agent that are used in the preparation of the formulation maydiffer from a final formulation. For example, some active agent may notbecome completely incorporated into a nanoparticle and such freetherapeutic agent may be e.g., filtered away. For example, in anembodiment, about 20 weight percent of active agent (e.g., docetaxel)and about 80 weight percent polymer (e.g., the polymer may include about2.5 mol percent PLA-PEG-GL2 and about 97.5 mol percent PLA-PEG). may beused in the preparation of a formulation that results in an e.g., finalnanoparticle comprising about 10 weight percent active agent (e.g.,docetaxel) and about 90 weight percent polymer (where the polymer mayinclude about 1.25 mol percent PLA-PEG-GL2 and about 98.75 mol percentPLA-PEG). Such processes may provide final nanoparticles suitable foradministration to a patient that includes about 2 to about 20 percent byweight therapeutic agent, e.g., about 5, about 8, about 10, about 15percent therapeutic agent by weight.

Lyophilized Pharmaceutical Compositions

Nanoparticles disclosed herein may be combined with pharmaceuticalacceptable carriers to form a pharmaceutical composition, according toanother aspect of the invention. As would be appreciated by one of skillin this art, the carriers may be chosen based on the route ofadministration as described below, the location of the target issue, thedrug being delivered, the time course of delivery of the drug, etc.

The pharmaceutical compositions of this invention can be administered toa patient by any means known in the art including oral and parenteralroutes. The term “patient,” as used herein, refers to humans as well asnon-humans, including, for example, mammals, birds, reptiles,amphibians, and fish. For instance, the non-humans may be mammals (e.g.,a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a primate,or a pig). In certain embodiments parenteral routes are desirable sincethey avoid contact with the digestive enzymes that are found in thealimentary canal. According to such embodiments, inventive compositionsmay be administered by injection (e.g., intravenous, subcutaneous orintramuscular, intraperitoneal injection), rectally, vaginally,topically (as by powders, creams, ointments, or drops), or by inhalation(as by sprays).

In a particular embodiment, the nanoparticles of the present inventionare administered to a subject in need thereof systemically, e.g.,parenterally, or by intravenous infusion or injection.

In some embodiments, a composition suitable for freezing iscontemplated, including nanoparticles disclosed herein and a solutionsuitable for freezing, e.g., a sugar such as a mono, di, or polysaccharide, e.g. sucrose and/or a trehalose, and/or a salt and/or acyclodextrin solution is added to the nanoparticle suspension. The sugar(e.g. sucrose or trehalose) may act, e.g., as a cryoprotectant toprevent the particles from aggregating upon freezing. For example,provided herein is a nanoparticle formulation comprising a plurality ofdisclosed nanoparticles, sucrose, an ionic halide, and water; whereinthe nanoparticles/sucrose/water/ionic halide is about3-40%/10-40%/20-95%/0.1-10% (w/w/w/w) or about 5-10%/10-15%/80-90%/1-10%(w/w/w/w). For example, such solution may include nanoparticles asdisclosed herein, about 5% to about 20% by weight sucrose and an ionichalide such as sodium chloride, in a concentration of about 10-100 mM.In another example, provided herein is a nanoparticle formulationcomprising a plurality of disclosed nanoparticles, trehalose,cyclodextrin, and water; wherein thenanoparticles/trehalose/water/cyclodextrin is about3-40%/1-25%/20-95%/1-25% (w/w/w/w) or about 5-10%/1-25%/80-90%/10-15%(w/w/w/w). For example, such solution may include nanoparticles asdisclosed herein, about 1% to about 25% by weight trehalose or sucrose(e.g. about 5% to about 25% trehalose or sucrose, e.g. about 10%trehalose or sucrose, or about 15% trehalose or sucrose, e.g. about 5%sucrose) by weight) and a cyclodextrin such as β-cyclodextrin, in aconcentration of about 1% to about 25% by weight (e.g. about 5% to about20%, e.g. 10% or about 20% by weight, or about 15% to about 20% byweight cyclodextrin). Comtemplated formulations may include a pluralityof disclosed nanoparticles (e.g. nanoparticles having PLA-PEG and anactive agent), and about 2% to about 15 wt % (or about 4% to about 6 wt%, e.g. about 5 wt %) sucrose and about 5 wt % to about 20% (e.g. about7% wt percent to about 12 wt %, e.g. about 10 wt %) HPbCD).

The present disclosure relates in part to lyophilized pharmaceuticalcompositions that, when reconstituted, has a minimal amount of largeaggregates. Such large aggregates may have a size greater than about 0.5μm, greater than about 1 μm, or greater than about 10 μm, and can beundesirable in a reconstituted solution. Aggregate sizes can be measuredusing a variety of techniques including those indicated in the U.S.Pharmacopeia at 32<788>, hereby incorporated by reference. The testsoutlined in USP 32<788> include a light obscuration particle count test,microscopic particle count test, laser diffraction, and single particleoptical sensing. In one embodiment, the particle size in a given sampleis measured using laser diffraction and/or single particle opticalsensing.

The USP 32<788> by light obscuration particle count test sets forthguidelines for sampling particle sizes in a suspension. For solutionswith less than or equal to 100 mL, the preparation complies with thetest if the average number of particles present does not exceed 6000 percontainer that are ≧10 μm and 600 per container that are ≧25 μm.

As outlined in USP 32<788>, the microscopic particle count test setsforth guidelines for determining particle amounts using a binocularmicroscope adjusted to 100±10× magnification having an ocularmicrometer. An ocular micrometer is a circular diameter graticule thatconsists of a circle divided into quadrants with black reference circlesdenoting 10 μm and 25 μm when viewed at 100× magnification. A linearscale is provided below the graticule. The number of particles withreference to 10 μm and 25 μm are visually tallied. For solutions withless than or equal to 100 mL, the preparation complies with the test ifthe average number of particles present does not exceed 3000 percontainer that are ≧10 μm and 300 per container that are ≧25 μm.

In some embodiments, a 10 mL aqueous sample of a disclosed compositionupon reconstitution comprises less than 600 particles per ml having asize greater than or equal to 10 microns; and/or less than 60 particlesper ml having a size greater than or equal to 25 microns.

Dynamic light scattering (DLS) may be used to measure particle size, butit relies on Brownian motion so the technique may not detect some largerparticles. Laser diffraction relies on differences in the index ofrefraction between the particle and the suspension media. The techniqueis capable of detecting particles at the sub-micron to millimeter range.Relatively small (e.g., about 1-5 weight %) amounts of larger particlescan be determined in nanoparticle suspensions. Single particle opticalsensing (SPOS) uses light obscuration of dilute suspensions to countindividual particles of about 0.5 μm. By knowing the particleconcentration of the measured sample, the weight percentage ofaggregates or the aggregate concentration (particles/mL) can becalculated.

Formation of aggregates can occur during lyophilization due to thedehydration of the surface of the particles. This dehydration can beavoided by using lyoprotectants, such as disaccharides, in thesuspension before lyophilization. Suitable disaccharides includesucrose, lactulose, lactose, maltose, trehalose, or cellobiose, and/ormixtures thereof. Other contemplated disaccharides include kojibiose,nigerose, isomaltose, β,β-trehalose, α,β-trehalose, sophorose,laminaribiose, gentiobiose, turanose, maltulose, palatinose,gentiobiulose, mannobiase, melibiose, melibiulose, rutinose, rutinulose,and xylobiose. Reconstitution shows equivalent DLS size distributionswhen compared to the starting suspension. However, laser diffraction candetect particles of >10 μm in size in some reconstituted solutions.Further, SPOS also may detect >10 μm sized particles at a concentrationabove that of the FDA guidelines (10⁴-10⁵ particles/mL for >10 μmparticles).

The present invention relates in part to the use of one or more ionichalide salts as an additional lyoprotectant to a sugar, such as sucrose,trehalose or mixtures thereof. Sugars may include disaccharides,monosaccharides, trisaccharides, and/or polysaccharides, and may includeother excipients, e.g. glycerol and/or surfactants. Optionally, acyclodextrin may be included as an additional lyoprotectant. Thecyclodextrin may be added in place of the ionic halide salt.Alternatively, the cyclodextrin may be added in addition to the ionichalide salt.

Suitable ionic halide salts may include sodium chloride, calciumchloride, zinc chloride, or mixtures thereof. Additional suitable ionichalide salts include potassium chloride, magnesium chloride, ammoniumchloride, sodium bromide, calcium bromide, zinc bromide, potassiumbromide, magnesium bromide, ammonium bromide, sodium iodide, calciumiodide, zinc iodide, potassium iodide, magnesium iodide, or ammoniumiodide, and/or mixtures thereof.

In one embodiment, about 1 to about 15 weight percent sucrose may beused with an ionic halide salt. In one embodiment, the lyophilizedpharmaceutical composition may comprise about 10 to about 100 mM sodiumchloride. In another embodiment, the lyophilized pharmaceuticalcomposition may comprise about 100 to about 500 mM of divalent ionicchloride salt, such as calcium chloride or zinc chloride. In yet anotherembodiment, the suspension to be lyophilized may further comprise acyclodextrin, for example, about 1 to about 25 weight percent ofcyclodextrin may be used.

Suitable cyclodextrin may include α-cyclodextrin, β-cyclodextrin,γ-cyclodextrin, or mixtures thereof. Exemplary cyclodextrinscontemplated for use in the compositions disclosed herein includehydroxypropyl-β-cyclodextrin (HPbCD), hydroxyethyl-β-cyclodextrin,sulfobutylether-β-cyclodextrin, methyl-β-cyclodextrin,dimethyl-β-cyclodextrin, carboxymethyl-β-cyclodextrin, carboxymethylethyl-β-cyclodextrin, diethyl-β-cyclodextrin,tri-O-alkyl-β-cyclodextrin, glocosyl-β-cyclodextrin, andmaltosyl-β-cyclodextrin. In one embodiment, about 1 to about 25 weightpercent trehalose (e.g. about 10% to about 15%, e.g. 5 to about 20% byweight) may be used with cyclodextrin. In one embodiment, thelyophilized pharmaceutical composition may comprise about 1 to about 25weight percent β-cyclodextrin. An exemplary composition may comprisenanoparticles comprising PLA-PEG, an active/therapeutic agent, about 4%to about 6% (e.g. about 5% wt percent) sucrose, and about 8 to about 12weight percent (e.g. about 10 wt. %) HPbCD.

In one aspect, the invention provides a lyophilized pharmaceuticalcomposition comprising polymeric nanoparticles, wherein uponreconstitution of the lyophilized pharmaceutical composition at ananoparticle concentration of about 50 mg/mL, in less than or about 100mL of an aqueous medium, the reconstituted composition suitable forparenteral administration comprises less than 6000, such as less than3000, microparticles of greater than or equal to 10 microns; and/or lessthan 600, such as less than 300, microparticles of greater than or equalto 25 microns.

The number of microparticles can be determined by means such as the USP32 <788> by light obscuration particle count test, the USP 32<788> bymicroscopic particle count test, laser diffraction, and single particleoptical sensing.

The nanoparticles may comprise a poly(lactic)acid-block-poly(ethylene)glycol copolymer orpoly(lactic)-co-poly(glycolic) acid-block-poly(ethylene)glycolcopolymer. For example, the poly(lactic) acid portion of the copolymermay have a weight average molecular weight of about 16 kDa and thepoly(ethylene)glycol portion of the copolymer may have a weight averagemolecular weight of about 5 kDa.

The reconstituted composition may have minimal aggregation compared to areconstituted composition that does not contain an ionic halide saltand/or a cyclodextrin. The reconstituted composition may have apolydispersity index of less than 0.2.

In an aspect, the invention provides a pharmaceutical compositionsuitable for parenteral use upon reconstitution, comprising a pluralityof therapeutic particles each comprising a copolymer having ahydrophobic polymer segment and a hydrophilic polymer segment; an activeagent; a sugar; and an ionic halide salt. The composition may furthercomprise a cyclodextrin.

The ionic halide salt may be selected from the group consisting ofsodium chloride, calcium chloride, and zinc chloride, or mixturesthereof. In an embodiment, the pharmaceutical composition may compriseabout 10 to about 100 mM sodium chloride. In another embodiment, thepharmaceutical composition may comprise about 100 to about 500 mMcalcium chloride or zinc chloride.

In an aspect, the invention provides a pharmaceutical compositionsuitable for parenteral use upon reconstitution, comprising a pluralityof therapeutic particles each comprising a copolymer having ahydrophobic polymer segment and a hydrophilic polymer segment; an activeagent; a sugar; and a cyclodextrin.

The cyclodextrin may include α-cyclodextrin, β-cyclodextrin,γ-cyclodextrin, or mixtures thereof. In an embodiment, thepharmaceutical composition may comprise about 1 to about 25 weightpercent β-cyclodextrin.

For example, the copolymer may be poly(lactic)acid-block-poly(ethylene)glycol copolymer. Upon reconstitution, a 100 mLaqueous sample may comprise less than 6000 particles having a sizegreater than or equal to 10 microns; and less than 600 particles havinga size greater than or equal to 25 microns.

In another aspect, the invention provides a pharmaceutically acceptableformulation for parenteral administration, prepared by a processcomprising: a) providing a composition comprising a plurality oftherapeutic particles each comprising a copolymer having a hydrophobicpolymer segment and a hydrophilic polymer segment; and an active agent;b) adding a disaccharide and an ionic halide salt to said composition;c) lyophilizing the composition to form a lyophilized composition; d)reconstituting the lyophilized composition to form the formulationsuitable for parenteral administration. In an embodiment, a cyclodextrinis included in the formulation. In some embodiments, such reconstitutingcan advantageously be managed with simple manual mixing for a fewminutes. The reconstituted product attributes (e.g. drug purity and/orrelease profile) may be substantially unchanged from a pre-lyophilizedcomposition (e.g. suspension).

In yet another aspect, the invention provides a pharmaceuticallyacceptable formulation for parenteral administration, prepared by aprocess comprising: a) providing a composition comprising a plurality oftherapeutic particles each comprising a copolymer having a hydrophobicpolymer segment and a hydrophilic polymer segment; and an active agent;b) adding a disaccharide and a cyclodextrin to said composition; c)lyophilizing the composition to form a lyophilized composition; d)reconstituting the lyophilized composition to form the formulationsuitable for parenteral administration. In some embodiments, suchreconstituting can advantageously be managed with simple manual mixingfor a few minutes. The reconstituted product attributes (e.g. drugpurity and/or release profile) may be substantially unchanged from apre-lyophilized composition (e.g. suspension).

The lyophilized composition may have a therapeutic particleconcentration of greater than about 40 mg/mL. The formulation suitablefor parenteral administration may have less than about 600 particleshaving a size greater than 10 microns in a 10 mL dose.

The step of adding a disaccharide and an ionic halide salt may compriseadding about 5 to about 15 weight percent sucrose or about 5 to about 20weight percent trehalose (e.g., about 10 to about 20 weight percenttrehalose), and about 10 to about 500 mM ionic halide salt. The ionichalide salt may be selected from sodium chloride, calcium chloride, andzinc chloride, or mixtures thereof. In an embodiment, about 1 to about25 weight percent cyclodextrin is also added.

In another embodiment, the step of adding a disaccharide and acyclodextrin may comprise adding about 5 to about 15 weight percentsucrose or about 5 to about 20 weight percent trehalose (e.g., about 10to about 20 weight percent trehalose), and about 1 to about 25 weightpercent cyclodextrin. In an embodiment, about 10 to about 15 weightpercent cyclodextrin is added. The cyclodextrin may be selected fromα-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or mixtures thereof.

The step of lyophilizing may comprise freezing the composition at atemperature of greater than about −40° C., or e.g. less than about −30°C., forming a frozen composition; and drying the frozen composition toform the lyophilized composition. The step of drying may occur at about50 mTorr at a temperature of about −25 to about −34° C., or about −30 toabout −34° C.

In another aspect, the invention provides a method of preventingsubstantial aggregation of particles in a pharmaceutical nanoparticlecomposition comprising adding a sugar and a salt to the lyophilizedformulation to prevent aggregation of the nanoparticles uponreconstitution. In an embodiment, a cyclodextrin is also added to thelyophilized formulation. In yet another aspect, the invention provides amethod of preventing substantial aggregation of particles in apharmaceutical nanoparticle composition comprising adding a sugar and acyclodextrin to the lyophilized formulation to prevent aggregation ofthe nanoparticles upon reconstitution.

EXAMPLES

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention inany way.

Example 1 Preparation of PLA-PEG

The synthesis is accomplished by ring opening polymerization ofd,l-lactide with α-hydroxy-ω-methoxypoly(ethylene glycol) as themacro-initiator, and performed at an elevated temperature using Tin (II)2-Ethyl hexanoate as a catalyst, as shown below (PEG Mn≈5,000 Da; PLAMn≈16,000 Da; PEG-PLA M_(n)≈21,000 Da).

The polymer is purified by dissolving the polymer in dichloromethane,and precipitating it in a mixture of hexane and diethyl ether. Thepolymer recovered from this step is dried in an oven.

Example 2 Exemplary Nanoparticle Preparation—Emulsion Process

An organic phase is formed composed of a mixture of docetaxel (DTXL) andpolymer (homopolymer, co-polymer, and co-polymer with ligand). Theorganic phase is mixed with an aqueous phase at approximately a 1:2ratio (oil phase:aqueous phase) where the aqueous phase is composed of asurfactant (0.25% sodium cholate) and some dissolved solvent (4% ethylacetate, 2% benzyl alcohol). In order to achieve high drug loading,about 30% solids in the organic phase is used.

The primary, coarse emulsion is formed by the combination of the twophases under simple mixing or through the use of a rotor statorhomogenizer. The rotor/stator yields a homogeneous milky solution, whilethe stir bar produces a visibly larger coarse emulsion. It is observedthat the stir bar method results in significant oil phase dropletsadhering to the side of the feed vessel, suggesting that while thecoarse emulsion size is not a process parameter critical to quality, itshould be made suitably fine in order to prevent yield loss or phaseseparation. Therefore the rotor stator is used as the standard method ofcoarse emulsion formation, although a high speed mixer may be suitableat a larger scale.

The primary emulsion is then formed into a fine emulsion through the useof a high pressure homogenizer. The size of the coarse emulsion does notsignificantly affect the particle size after successive passes (103)through the homogenizer.

After 2-3 passes the particle size is not significantly reduced, andsuccessive passes can even cause a particle size increase. The organicphase is emulsified 5:1 O:W with standard aqueous phase, and multiplediscreet passes are performed, quenching a small portion of emulsionafter each pass. The indicated scale represents the total solids of theformulation.

The effect of scale on particle size shows scale dependence. The trendshows that in the 2-10 g batch size range, larger batches producesmaller particles. It has been demonstrated that this scale dependenceis eliminated when considering greater than 10 g scale batches. Theamount of solids used in the oil phase is about 30%.

Table A summarizes the emulsification process parameters.

TABLE A Parameter Value Coarse emulsion formation High shear mixerHomogenizer feed pressure 2500 psi per chamber Interaction chamber(s) 4× 200 μm Z-chamber Number of homogenizer passes 1 pass Water phase0.25-0.35% [sodium cholate] W:O ratio 2:1 [Solids] in oil phase 30%

The fine emulsion is then quenched by addition to deionized water at agiven temperature under mixing. In the quench unit operation, theemulsion is added to a cold aqueous quench under agitation. This servesto extract a significant portion of the oil phase solvents, effectivelyhardening the nanoparticles for downstream filtration. Chilling thequench significantly improves drug encapsulation. The quench:emulsionratio is approximately 5:1.

A solution of 35% (wt %) of Tween 80 is added to the quench to achieveapproximately 4% Tween 80 overall After the emulsion is quenched asolution of Tween-80 is added which acts as a drug solubilizer, allowingfor effective removal of unencapsulated drug during filtration. Table Bindicates each of the quench process parameters.

TABLE B Summary quench process parameters. Parameter Value Initialquench temperature <5° C. [Tween-80] solution 35% Tween-80:drug ratio25:1 Q:E ratio 10:1 Quench hold/ ≦5° C. (with processing temp current5:1 Q:E ratio, 25:1 Tween- 80:drug ratio)

The temperature must remain cold enough with a dilute enough suspension(low enough concentration of solvents) to remain below the T_(g) of theparticles. If the Q:E ratio is not high enough, then the higherconcentration of solvent plasticizes the particles and allows for drugleakage. Conversely, colder temperatures allow for high drugencapsulation at low Q:E ratios (to ˜3:1), making it possible to run theprocess more efficiently.

The nanoparticles are then isolated through a tangential flow filtrationprocess to concentrate the nanoparticle suspension and buffer exchangethe solvents, free drug, and drug solubilizer from the quench solutioninto water. A regenerated cellulose membrane is used with a molecularweight cutoffs (MWCO) of 300.

A constant volume diafiltration (DF) is performed to remove the quenchsolvents, free drug and Tween-80. To perform a constant-volume DF,buffer is added to the retentate vessel at the same rate the filtrate isremoved. The process parameters for the TFF operations are summarized inTable C. Crossflow rate refers to the rate of the solution flow throughthe feed channels and across the membrane. This flow provides the forceto sweep away molecules that can foul the membrane and restrict filtrateflow. The transmembrane pressure is the force that drives the permeablemolecules through the membrane.

TABLE C TFF Parameters Parameter Optimized Value Membrane Regeneratedcellulose—Coarse Material Screen Membrane Molecular Weight 300 kDa Cutoff Crossflow Rate 3.7-10 L/min/m² Transmembrane ~5 psid PressureConcentration of 30-50 mg/ml Nanoparticle Suspension for DiafiltrationNumber of 20) Diavolumes Membrane Area 5 m²/kg

The filtered nanoparticle slurry is then thermal cycled to an elevatedtemperature during workup. A small portion (typically 5-10%) of theencapsulated drug is released from the nanoparticles very quickly afterits first exposure to 25° C. Because of this phenomenon, batches thatare held cold during the entire workup are susceptible to free drug ordrug crystals forming during delivery or any portion of unfrozenstorage. By exposing the nanoparticle slurry to elevated temperatureduring workup, this ‘loosely encapsulated’ drug can be removed andimprove the product stability at the expense of a small drop in drugloading. Table D summarizes two examples of 25° C. processing. Otherexperiments have shown that the product is stable enough after ˜2-4diavolumes to expose it to 25° C. without losing the majority of theencapsulated drug. 5 diavolumes is used as the amount for coldprocessing prior to the 25° C. treatment.

TABLE D Lots A Lots B Drug load Cold workup 11.3% 9.7% 25° C. workup¹8.7-9.1% 9.0-9.9% Stability² Cold workup <1 day <1 day 25° C. workup¹5-7 days 2-7 days In vitro burst³ Cold workup ~10% Not 25° C. workup¹ ~2% performed ¹25° C. workup sublots were exposed to 25° C. after atleast 5 diavolumes for various periods of time. Ranges are reportedbecause there were multiple sublots with 25° C. exposure. ²Stabilitydata represents the time that final product could be held at 25° C. at10-50 mg/ml nanoparticle concentrations prior to crystals forming in theslurry (visible by microscopy) ³In vitro burst represents the drugreleased at the first time point (essentially immediately)

After the filtration process, the nanoparticle suspension is passedthrough a sterilizing grade filter (0.2 μm absolute). Pre-filters areused to protect the sterilizing grade filter in order to use areasonable filtration area/time for the process. Values are assummarized in Table E.

TABLE E Parameter O Value Effect Nanoparticle 50 mg/ml Yield losses arehigher at higher [NP], but the Suspension ability to filter at 50 mg/mlobviates the need to Concentration aseptically concentrate afterfiltration Filtration flow ~1.3 Filterability decreases as flow rateincreases rate L/min/m²

The pre-filter has Seitz PDD1 depth filter media in Pall SUPRAcap orStax filter cartridges. 0.2 m² of filtration surface area per kg ofnanoparticles for depth filters and 1.3 m² of filtration surface areaper kg of nanoparticles for the sterilizing grade filters can be used.

Example 3 Lyophilized Composition With Sugar and Salt

As shown in FIG. 3, nanoparticle suspensions with >40 mg/mL nanoparticleconcentrations (with nanoparticles formed as in Example 2, with 16/5PLA-PEG as the polymer) are lyophilized in the presence of 10% sucroseand an additive: NaCl, CaCl₂, or PBS. This experiment formulatesnanoparticle suspensions at high (>40 mg/ml) nanoparticle concentrationsthat can be reconstituted without micro-aggregation. All three CaCl₂formulations produce reconstituted cakes with <100 particles/ml (10μm+), even in the mid (150 mM) and high (200 mM) concentration rangeswhich produced a lyophilisate that had collapsed.

Lower concentrations of salt behave similarly as in the absence of salt.Higher concentrations of salt generally show much higher particleconcentrations.

Example 4 Lyophilized Composition with Sugar and/or Salt and/orCyclodextrin

Nanoparticle suspensions are lyophilized in the presence of a sugar(e.g. sucrose or trehalose), salt (e.g. NaCl or CaCl₂), and/orcyclodextrin (e.g. hydroxypropyl beta cyclodextrin—HPbCD). For example,formulations are prepared with 250 mM or 500 mM of NaCl or CaCl₂; and/orwith 15%, 20% or 25% by weight sucrose or trehalose, for example, 20% byweight trehalose, 500 mM CaCl₂, 5% HPbCd. Representative formulationsare shown in Table F.

Table F indicates particles counted and sized one at a time over a largesize range by an AccuSizer, and counted the larger size of particlenumbers to find the aggregates that existed in formulation. Table Fshows the number of particles after reconstituting solution usingdistilled water into lyophilized cakes. In the Table F, F/T samples arefrom freezing and thawing only without drying, whereas vial number from1 to 4 as well as tall vial 1 and 2 were from lyophilized samples. Mostof formulations except CaCl₂ 500 mM with 15% Trehalose showed low numberof particle aggregates and subsequent tests were done to optimize theformulations.

TABLE F Particle Number/ml (larger than 10 μm) F/T Tall vial Tall VialFormulation Reconstitution control 1 2 3 4 1 2 CaC1₂ + 15% Good 282 527333 940 396 1110 430 sucrose CaC1₂ + 15% Dissolved right 310 17600 548Vial 1160000 1190 442 trehalose away broke CaC1₂ + 20% quick 945 446 670486 3500 384 713 trehalose + 5% HPbCD 20% quick 392 28300 4210 899 2790239 75.5 trehalose + 10% HPbCD

Example 5 Lyophilized Composition With Sugar and/or Salt and/orCyclodextrin

Nanoparticle suspensions are lyophilized in the presence of a sugar(e.g. trehalose), cyclodextrin (e.g. hydroxypropyl betacyclodextrin—HPbCD), and/or salt (e.g. CaCl₂). The excipient and levelto screen formulations using Design of Experiment (DOE) are listed belowin Table G. Tall vials are used for all formulations with a fill volumeof 5 mL (n=5-6 vials per formulation). Primary drying is performed at−37° C. shelf temperature.

TABLE G Excipient Level 1 Level 2 Level 3 Level 4 HPbCD  5% 10% N/A N/ATrehalose 10% 20% N/A N/A CaCl₂ 0 mM 100 mM 250 mM 500 mM

The appearance of the lyophilized formulations and their reconstitutionproperties are listed below in Table H. In all the formulations tested,the appearance of the formulations is at least partially melted.

TABLE H Appearance Post Formulation              

  Lyophilization  

  Reconstitution      

   5% HPbCD partially melted back OK, very turbid 10% HPbCD partiallymelted back OK 10% Trehalose + 10% HPbCD partially melted back Requiredlots of vortexing 10% Trehalose + 5% HPbCD partially melted backRequired lots of vortexing 20% Trehalose + 10% HPbCD partially meltedback Required lots of vortexing 20% Trehalose + 5% HPbCD partiallymelted back No, small chunks remain CaCl2 100 mM + 10% Trehalose + 10%HPbCD partially melted back Required lots of vortexing CaCl2 100 mM +10% Trehalose + 5% HPbCD partially melted back Required lots ofvortexing CaCl2 100 mM + 20% Trehalose + 10% HPbCD partially melted backRequired tons of vortexing CaCl2 100 mM + 20% Trehalose + 5% HPbCD B&Ecompletely NO, large chunks remain collapsed; A/C/D partially CaCl2 250mM + 10% Trehalose + 10% HPbCD partially melted back Required lots ofvortexing CaCl2 250 mM + 10% Trehalose + 5% HPbCD A collapsed; othersYes, with no mixing partially melted back CaCl2 250 mM + 20% Trehalose +10% HPbCD partially melted back Required lots of vortexing CaCl2 250mM + 20% Trehalose + 5% HPbCD partially melted back Required tons ofvortexing CaCl2 500 mM + 10% Trehalose + 10% HPbCD partially melted backRequired tons of vortexing CaCl2 500 mM + 10% Trehalose + 5% HPbCDMostly collapsed No—Required tons of vortexing AND time CaCl2 500 mM +20% Trehalose + 10% HPbCD Mostly collapsed Required tons of vortexingCaCl2 500 mM + 20% Trehalose + 5% HPbCD Mostly collapsed and Requiredtons of vortexing AND partially blown up time

Cycle data is shown in FIG. 4, and shows lyophilization processparameters: shelf temperature, product temperature, chamber pressure andtime. These process parameters are controlled from the time the productis first placed on the lyophilizer shelves during loading until theproduct is removed. Conditions reflected in the chart illustrate theprocess parameters for one of respective lyo run to screen HPbCDconcentration.

The sizes of the particles in the various lyophilized formulations aremeasured by dynamic light scattering (DLS) and shown in FIG. 5. In allthe formulations tested, the nanoparticle size increased afterfreeze/thaw and lyophilization as compared to pre-frozen samples.

The number of microparticles greater than 10 μm in the variousformulations are measured by microscopic particle count test and shownin FIG. 6. In general, formulations comprising higher concentrations ofcyclodextrin exhibit better particulate counts.

Example 5 Lyophilized Composition With Sugar and Cyclodextrin

Nanoparticle suspensions are lyophilized in the presence of a sugar(e.g. trehalose or sucrose) and cyclodextrin (e.g. hydroxypropyl betacyclodextrin—HPbCD). The formulations tested are listed below in TableH. Tall vials are used for all formulations with a fill volume of 5 mL(n=10 vials per formulation).

TABLE H Excipient Level 1 Level 2 Level 3 Level 4 HPbCD 10% 15% 20% NATrehalose  0%  5% 10% 20% Alternative Variable Levels Formulation(s)Sugar Type Sucrose 1) 10% HPbCD, 10% Sucrose 2) 10% HPbCD, 5% Sucrose

The appearance of the lyophilized formulations and their reconstitutionproperties are listed below in Table I. Increased concentration oftrehalose and cyclodextrin appeared to result in poorer reconstitutionproperties. In all the formulations tested, the DLS sizes increasedafter freeze/thaw but decreased after lyophilization.

TABLE I DXTL Conc Appearance Post Formulation      

  (mg/ml 

  Lyophilization 

  Reconstitution            

   0% Trehalose, 10% HPbCD 4.803 partially melted back ok—manual mixingrequired (<1 min)  0% Trehalose, 15% HPbCD 4.711 partially melted backok—manual mixing required (<1 min ea)— some lg chunks to disperse  0%Trehalose, 20% HPbCD 4.736 partially melted back Lots of manual mixingrequired (couple of min)  5% Trehalose, 10% HPbCD 4.328 partially meltedback majority reconstituted immediate but some sm chunks neededadditional mixing  5% Trehalose, 15% HPbCD 4.674 partially melted backsome reconstituted immediately but more chunks than −4 which neededextra mixing  5% Trehalose, 20% HPbCD 4.23 partially melted backmajority reconstituted immediate but some sm chunks needed additionalmixing 10% Trehalose, 10% HPbCD 4.28 partially melted back majorityreconstituted immediate, a bit of extra mixing 10% Trehalose, 15% HPbCD4.637 partially melted back majority reconstituted immediate, a bit ofextra mixing 10% Trehalose, 20% HPbCD 4.158 partially melted back somereconstituted quickly but had to do extra mixing to get chunks in 20%Trehalose, 10% HPbCD 3.655 partially melted back Had chunks butreconstitued with shaking 20% Trehalose, 15% HPbCD 3.397 partiallymelted back Had chunks but reconstitued with 1.5 min shaking 20%Trehalose, 20% HPbCD 4.392 partially melted back Had chunks butreconstitued with 2 min shaking  5% Sucrose, 10% HPbCD 4.614 partiallymelted back reconstituted ok within ½ min shaking (probably less) 10%Sucrose, 10% HPbCD 4.686 partially melted back reconstituted ok within ½min shaking (probably less)

Cycle data is shown in FIG. 7. The size of the particles in the variouslyophilized formulations are measured by dynamic light scattering (DLS)and shown in FIG. 8.

The number of microparticles per ml which are greater than 10 μm in thevarious formulations are measured by microscopic particle count test asshown in FIG. 9. Almost all the formulations tested are below the USP32<788> limit. The number of microparticles which are greater than 1 μmin the various formulations are shown in FIGS. 10 and 11. In mostformulations, the number of microparticles greater than 1 μm isincreased in the lyophilized samples when compared to the pre-frozen orfreeze/thaw samples.

In vitro release test is performed on docetaxel nanoparticleslyophilized in the presence of sugar and cyclodextrin. Results aredepicted in FIG. 12.

Differential scanning calorimetry is also performed on variousnanoparticle formulations as depicted in FIGS. 13-16.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

INCORPORATION BY REFERENCE

The entire contents of all patents, published patent applications,websites, and other references cited herein are hereby expresslyincorporated herein in their entireties by reference.

1. A method of preparing a reconstituted lyophilized pharmaceuticalcomposition suitable for parenteral administration, comprising:providing a formulation comprising polymeric nanoparticles, wherein thepolymeric nanoparticles comprise a therapeutic agent and a polymerselected from the group consisting of poly(lactic)acid-block-poly(ethylene)glycol copolymer andpoly(lactic)-co-poly(glycolic) acid-block-poly(ethylene)glycolcopolymer; adding hydroxypropyl β-cyclodextrin and a sugar selected fromthe group consisting of trehalose and sucrose to the formulation suchthat the formulation has 7 to 12 weight percent of the hydroxypropylβ-cyclodextrin and 4 to 6 weight percent of the sugar; lyophilizing theformulation to foil a lyophilized formulation; and reconstituting thelyophilized formulation in an aqueous medium to form the reconstitutedlyophilized pharmaceutical composition, wherein the reconstitutedlyophilized pharmaceutical composition has a concentration of 10-100mg/mL of the polymeric nanoparticles.
 2. The method of claim 1, whereinthe sugar is trehalose.
 3. The method of claim 1, wherein the sugar issucrose.
 4. The method of claim 1, wherein the reconstituted lyophilizedpharmaceutical composition comprises less than 6000 microparticles ofgreater than or equal to 10 microns; and less than 600 microparticles ofgreater than or equal to 25 microns; per sample container having lessthan or about 100 mL of the composition.
 5. The method of claim 1,wherein the reconstituted lyophilized pharmaceutical compositioncomprises less than 3000 microparticles having a size greater than orequal to 10 microns; and less than 300 microparticles having a sizegreater than or equal to 25 microns; per sample container having lessthan or about 100 mL of the composition.
 6. The method of claim 1,wherein the reconstituted lyophilized pharmaceutical composition hasminimal aggregation compared to a reconstituted lyophilizedpharmaceutical composition that does not contain a cyclodextrin.
 7. Themethod of claim 1, wherein the polymer is poly(lactic)acid-block-poly(ethylene)glycol copolymer.
 8. The method of claim 7,wherein the poly(lactic) acid-block-poly(ethylene)glycol copolymercomprises poly(lactic acid) having a number average molecular weight ofabout 15 to about 20 kDa and poly(ethylene)glycol having a numberaverage molecular weight of about 4 to about 6 kDa.
 9. The method ofclaim 8, wherein the poly(lactic acid) has a number average molecularweight of about 16 kDa and the poly(ethylene)glycol has a number averagemolecular weight of about 5 kDa.
 10. The method of claim 1, wherein thepolymer is poly(lactic)-co-poly(glycolic)acid-block-poly(ethylene)glycol copolymer.
 11. The method of claim 1,wherein the therapeutic agent is docetaxel.
 12. The method of claim 1,wherein the reconstituted lyophilized pharmaceutical compositioncomprises about 40-60 mg/mL concentration of polymeric nanoparticles.13. The method of claim 1, wherein the polymeric nanoparticles have adiameter of about 60 nm to about 140 nm.
 14. A method of preparing apharmaceutically acceptable aqueous solution, comprising: providing aformulation comprising polymeric nanoparticles, wherein the polymericnanoparticles comprise a therapeutic agent and a polymer selected fromthe group consisting of poly(lactic) acid-block-poly(ethylene)glycolcopolymer and poly(lactic)-co-poly(glycolic)acid-block-poly(ethylene)glycol copolymer; adding hydroxypropylβ-cyclodextrin and sucrose to the formulation such that the formulationhas 7 to 12 weight percent of the hydroxypropyl β-cyclodextrin and 4 to6 weight percent of the sucrose; lyophilizing the formulation to form alyophilized formulation; and reconstituting the lyophilized formulationin an aqueous medium to form the pharmaceutically acceptable aqueoussolution, wherein the pharmaceutically acceptable aqueous solution has aconcentration of 40-60 mg/mL of the polymeric nanoparticles.